How the COX2 Promoter’s Unique Sequence Affects the First Step of Mitochondrial Transcription Initiation

• Isabella Villa, Biochemistry, University of Delaware

• Jeremy Bird, College of Arts & Sciences, University of Delaware

DNA transcription initiation is the beginning of the flow of genetic information in the cell towards gene expression, and many labs have worked towards understanding its mechanisms. The basic stages of transcription initiation include, binding promoter DNA to transcription factors and RNA polymerase, unwinding the DNA to form the “transcription bubble”, and finally recruiting nucleotides to synthesize RNA. Transcription initiation of mitochondrial DNA (mtDNA), while similar to eukaryotic nuclear transcription, has some key differences. Mitochondrial transcriptional components are simpler, using only one transcription factor and a single-protein RNA polymerase, unlike nuclear transcription, which requires many transcription factors and a multi-protein polymerase. This project investigates the roles of mitochondrial RNA polymerase (Rpo41) and mitochondrial transcription factor 1 (Mtf1) in mtDNA transcription initiation of Saccharomyces cerevisiae (Brewers yeast). We examined the first step of transcription initiation; promoter binding. We measured binding affinity and specificity of Mtf1 and Rpo41 to variants of the COX2 promoter sequence using Electrophoresis Mobility Shift Assays (EMSAs). COX2 is the only mitochondrial promoter that naturally has an A/T base pair at position -5 of the initiation sequence. To test the significance of this base, we designed four yeast mtDNA templates, each containing a different nucleotide at the -5 position. By comparing binding across these variants, we explored how a single nucleotide change affects protein-DNA interactions during transcription initiation. Based on the results it’s clear the position -5 base pair is important for promoter binding because DNA binding didn’t occur with all four promoter variants. Ultimately these findings can contribute to a larger understanding of the detailed mechanisms of yeast mtDNA transcription initiation which can tell us more about mitochondrial gene expression. Lastly, these proteins and DNA have homologous structures in human mitochondria so this research can also be useful in deciphering the mechanisms of our own mitochondria.